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Abstract

Aim: Mesenchymal stem cells are an excellent potential source of cells for bone tissue engineering due to their excellent renewal ability and osteogenic differentiation capabilities. This study was designed to evaluate the bone formation properties of a demineralized cancellous bone scaffold seeded with mesenchymal stem cells, with or without periosteum, in a critical size bone defect model in rabbits.

Results: New bone formation and bone healing were successfully achieved, both radiologically and histologically, on demineralized cancellous bone graft seeded with BM-MSCs. Results were improved when BM-MSCs were associated with periosteum.

Conclusion: This study demonstrates that repair of bone defects in a rabbit model can be achieved through bone grafting using bone marrow mesenchymal stem cells implanted on a demineralized cancellous bone scaffold. The formation of new bone was optimized when combined with the preservation of periosteum at the site of injury.

Introduction

Despite numerous advances in orthopaedic and plastic surgery, the repair of bone defects remains challenging. The most desirable material for bone repair is autologous bone graft, due to its excellent osteoconduction, osteoinduction, and osteogenesis properties.[1,2] However, its limitations include additional surgical exposure required for graft harvest, limited bone supply, and associated donor site morbidities.[3,4] Vascularized bone grafts from various locations including the fibula, scapula, and iliac crest may be indicated to stimulate bone formation and promote healing. However, harvest requires a complex microsurgery procedure, with the additional risk of including graft necrosis due to vessel thrombosis.[5,6] Allografts may be a reasonable alternative, as small cancellous allografts can remodel completely. Larger grafts may incorporate by limited intramembranous bone formation.[1] However, allograft may increase the risk of infectious disease transmission.

Recent progress in the fields of biotechnology and tissue engineering has offered new options for repair of traumatic and non-traumatic bone defects. Mesenchymal stem cells (MSCs), which are multipotent adult stem cells of mesodermal origin, have been shown to play a critical role in tissue engineering. MSCs are an excellent potential source of cells for bone tissue engineering due to their excellent renewal ability and osteogenic differentiation capabilities.[7,8] In addition to the bone marrow, MSCs are also derived from the periosteum. It is well known that the development and regeneration of bone depend on the presence of periosteum and bone marrow.[9] When transferred to the site of bone damage, MSCs multiply and differentiate into osteoblastic cells, contributing to the production of bone tissues that form a callus at the bone defect site.[10] Alternatively, bone tissue engineering can be achieved via intramembranous ossification.[11]

The use of MSCs with an appropriate scaffold has been demonstrated to be promising in guiding bone tissue neoformation after implantation in the host. Cell repopulation can be achieved either by direct cell loading or indirect cell induction with osteogenic factors.[12,13] Combining MSCs with appropriate scaffolds has been shown to improve the overall osteoconductivity of the scaffold. The search for an ideal scaffold has led to the development of reconstructive options to engineer new bone tissue. The ideal scaffold should be biocompatible, non-infectious, resorbable, osteoconductive, and osteoinductive.[14] Demineralized bone matrix (DBM), which is derived from either allogenic or xenogenic bone, is available commercially for clinical application and satisfies some of these requirements.[4] DBM has been used for several decades in humans for the treatment of nonunion and bone defects following injury or tumor resection. The process of demineralization using hydrochloric acid destroys potential bone forming agents, but also decreases antigenic stimulation and may expose the bone morphogenic protein located within the bone matrix.[1,4] This study is designed to evaluate the bone formation properties of a demineralized cancellous bone scaffold seeded with allogenic MSCs, with or without periosteum, in a critical sized bone defect model in rabbits.

Human demineralized cancellous bone (HDCB), which is a type of DBM and has been proven to be usable as scaffold material, was used in this experiment. HDCB was supplied from the Bone Bank at the National Institute of Burns. Fresh bones were aseptically harvested within the first 12 h after being shown to be free of any infectious disease. Bones were treated with H2O2, a mixture of methanol/chloroform, hydrochloric acid, and phosphate buffer pH 7.4. Subsequently the bones were dehydrated for 24 h until the water content remaining in the bones was less than 5%. The bones were cut into blocks with dimensions of 1.5 cm × 0.3 cm × 0.5 cm. A medullary hole was made in the bone blocks with a diameter of 1.5 mm. The block was packaged and sterilized by gamma irradiation at a dose of 25 kGy. The sterilized bones were then preserved at 4℃.

Tissue engineered bone graft preparation in vitro

Culture-expanded BM-MSCs were seeded evenly onto the HDCB scaffold. DHCB/BM-MSCs were cultured in T flasks (Thermo Scientific Nunc A/S, Denmark) filled with 5mL DMEM containing 10% FBS and antibiotics. The grafts were placed in a vacuum desiccator and treated at a pressure of 100 Torr for 100 seconds, after which they were incubated at 37℃, 5% CO2 for 2 weeks. The medium was replaced every 3 days.[2,19-21]

Animals and surgical procedure

Twenty-eight males 8-week-old New Zealand white rabbits with a body weight of approximately 1.5 kg from the Experimental Laboratory of the Medical Learning and Research Center, Hue Central Hospital, were used for the study. The protocol for this experimental study was approved by the Committee of the Medical Learning and Research Center. The rabbit bone defect model was established as described previously.[11,13] The rabbit was anesthetized with a combination of intravenous sodium pentobarbital at 20 mg/kg and intramuscular ketamine at 50 mg/kg. The anterolateral side of the forelimb was shaved and sterilized with 10% povidone-iodine. The radius was exposed through a longitudinal incision by gentle retraction of the muscles. An osteotomy gap of 1.5 cm was created in the diaphysis. Periosteum from the excised bone was preserved in the group that would later receive periosteal encapsulation of scaffolds. The ulna was left intact for mechanical stability [Figure 1]. The bone defect was created on both forelimbs of the animals. A total of 56 bone defects within the 28 rabbits were randomly assigned to one of four groups for scaffold implantation: group 1: HDCB graft only; group 2: periosteum-wrapped HDCB graft; group 3: HDCB graft seeded with BM-MSCs; and group 4: periosteum-wrapped HDCB graft seeded with BM-MSCs. After implantation, muscle, fascia and skin were separately closed over the defect and no internal or external fixation was used. Forelimbs were post-operatively supported by a carton splint for one week. Each rabbit was administered 400,000 units of penicillin preoperatively and the first postoperative day to prevent infection. All rabbits from each group were sacrificed 12 weeks after surgery for gross observation of the growth of callus, radiological assessment, histological analyses, and biomechanical measurements.

Figure 1: The procedure for the transplantation of cancellous bone graft into the segmental radial defect

Following sacrifice, both reconstructed radiuses were harvested and completely cleared from the soft tissues. The status of callus growth, degradation, bone healing, and new bone formation at the bone graft in the radius were observed.

Radiological assessment

Radius bone specimens in each group were X-rayed for evaluation of bone formation and remodeling (Titan 2000, COMED Medical Systems CO. Ltd., Korea). Assessment of new bone formation and remodeling was based on the modified Lane and Sandhu radiological scoring system.[1] Three experts blindly assessed radiological scores, which were the sum of the scores of bone formation and remodeling. The score for new bone formation was assigned as 0 (no new bone formation), 1 (< 25% new bone formation), 2 (25-50% new bone formation), 3 (50-75% new bone formation), or 4 (> 75% new bone formation). The score assigned to the assessment of union was 0 (nonunion), 1 (possible union), or 2 (radiographic union). The proximal and distal unions of the bone graft were separately evaluated. The remodeling score assigned was 0 (no evidence of remodeling), 2 (intramedullary remodeling), or 4 (cortical remodeling). The maximum number of points, which could be achieved, was 10 for each reconstructed bone.

Histological analyses

Fifty-two specimens from the bone graft sites of the radius were successfully fixed with 10% paraformaldehyde, decalcified with sodium formate and embedded in paraffin. Four specimens in group 1 experienced technical failures. Three sagittal sections were cut with a slow speed saw from each site at the distal, proximal and middle lines of the bone graft. Sections were then prepared and stained with hematoxylin and eosin. The micrographic images from the light microscope were quantified. Images from each section were taken to evaluate the bone formation ratio by a qualified pathologist blinded to the study. The new bone formation ratio was calculated by the percentage area of bone tissue within the defect site, and a mean value was determined for each section.

Biomechanical analysis

The specimens of the radius of each group were loaded onto a multifunctional mechanical tester (Instron 5582 Universal Tester, USA) for performance of a uniaxial compression test. The specimen was placed between compression plates. Force was applied to the specimens at a constant speed of 1 mm/min until fracture occurred. Compressive stress and strain were calculated and plotted. Stress value at the point of yield (load-to-failure) was determined.

Statistical analysis: The data were presented as mean and standard deviation. The Student’s test was performed to compare the difference between the mean values of 2 groups using Statistical Product and Service Solutions (SPSS) version 15.0 (SPSS Inc. USA). Differences at a level of P value of less than 0.05 were considered to be statistically significant.

Results

The wounds healed completely after one week and the rabbits were noted to regain full movement within two weeks. All rabbits survived with normal behavior. No complications such as infection or necrosis were recorded prior to sacrifice.

Gross observation

At 12 weeks after surgery, radii implanted in group 1 showed a small amount of callus and fibrous-like tissue in the interspaces between the defects and grafts. Partial degradation of the HDCB grafts was found. There was a significant amount of callus and bony union filled more than half of the defects in groups 2 and 3. The HDCB grafts in these groups were almost degraded. In group 4, good bony union was observed. Bone defects were almost completely remodeled with new bone tissue and the HDCB grafts were completely degraded in this group [Figure 2].

Figure 2: Gross observations of the reconstruction of radius at 3 months after surgery. (a) Small amount of callus and fibrous‑like tissue in the interspaces between defect and human demineralized cancellous bone graft in Group 1; (b) callus formed in the defect repair by periosteum‑wrapped human demineralized cancellous bone graft in Group 2; (c) significant amount of callus and bony union filled in the defect repair with the human demineralized cancellous bone graft seeded with mesenchymal stem cells in Group 3; (d) complete bone healing in the defect repair by periosteum‑wrapped human demineralized cancellous bone graft seeded with bone marrow mesenchymal stem cells in Group 4

At 3 months postoperatively, there were a small amount of callus formation at the defect gaps in group 1. New bone formation was found to account for over half of the material at the reconstructed bone in groups 2 and 3. Bone regeneration in the radius in group 4 was observed to be the best, where callus formation was greatest in comparison to the other groups [Figure 3]. With the radiological score results, the mean score in group 4 was 8.58 ± 0.64, which was statistically significantly higher than that in the other three groups (P < 0.05). There was significant difference between groups 2 and 3 (P < 0.05). The mean scores in groups 2 and 3 were significantly higher than those in group 1 (P < 0.05) [Table 1].

Table 1: Modified Lane and Sandhu radiological scores, mean new bone formation in Histology (%), and mean compressive strength (MPa) of the rabbit's radius in each group at 3 months after surgery

Figure 3: Results of X‑ray at the 3 months postoperation. (a) A few calluses at the defect gap in Group 1; (b) significant new bone information at the reconstructed bone in Group 2; (c) more new bone formation between graft and bone tissue in Group 3; (d) almost remodeling of new formed bone along the entire gap of the bone defect in Group 4, and the cortical bone bridged to the adjacent native bone

Inflammation was not observed in the grafted bone segment. Poor new bone formation and capillary network were found at the interface between the graft and radius in group 1. Both ends of the original radius were united with newly regenerated bone in groups 2 and 3, while the HDCB scaffold was mostly degraded and cortical bone was only observed at the center of the defects. A larger amount of new bone was generated along the entire scaffold structure and more capillaries were formed in the area of new bone in group 4. Group 4 showed superior bone union, cancellous bone, cortical bone, marrow formation, and capillary formation in comparison to the other groups. Cortical bone was also found along the entire gap of the bone defect bridging adjacent native bone [Figure 4]. The newly formed bone area in group 4 increased to 80.5% ± 4.96%, which was significantly higher when compared with group 3 (64.12% ± 11.31%), group 2 (49.79% ± 11.69%) and group 1 (29.6% ± 8.33%) [Table 1] (P < 0.05). Statistically significant differences were found between groups 2 and 3 (P < 0.05), while both groups were statistically superior as compared to group 1 (P < 0.05) [Table 1].

Radii of rabbits with partial or complete union were subjected to biomechanical testing. Results of the biomechanical tests are summarized in Table 1. Group 4 showed the highest compressive strength (P < 0.05). Group 3 of HDCB grafts seeded with BM-MSCs showed significantly higher compressive strength than both groups 1 and 2 (P < 0.05). The difference between groups 1 and 2 was statistically significant (P < 0.05) [Table 1].

Discussion

This study demonstrates the presence of new bone formation and bone healing, as shown both radiologically and histologically, on demineralized cancellous bone graft seeded with BM-MSCs. Results were improved when BM-MSCs were associated with periosteum.

Mesenchymal stem cells, periosteal cells and osteoblasts have all been successfully used for bone tissue engineering.[4,18] In particular, bone marrow mesenchymal stem cells (BM-MSCs) play a major role in the repair of bone defects.[22-25] They are capable of self-replication and differentiation into osteocytes in appropriate culture conditions, and can contribute to the regeneration of mesenchymal tissues such as bone.[3,26] BM-MSCs can be rapidly expanded ex vivo without loss of their multi-lineage differentiation potential.[13] They are readily available and amenable to genetic manipulation. BM-MSCs can therefore be viewed as a viable alternative for bone tissue engineering.[8,11,27,28]

The anatomy of the periosteum, its nutrient transport, and its osteoinductive and osteoconductive capacities have been well described.[29] Periosteum plays a primary role in bridging callus formation and participating in endochondral and intramembranous ossifications in fracture healing.[30] Previous studies have shown that the inner cambium layer is highly cellular and populated with cells which influence bone formation and bone repair, including adult mesenchymal skeletal progenitor cells.[29,31] These progenitor cells proliferate and differentiate into osteoblastic and chondroblastic cells, driving the process of bone repair via either direct intramembranous bone formation or indirect endochondral mechanisms, respectively.[32] On the contrary, the absence of periosteum reduced by 75% the number of osteoblasts on devitalized bone graft, which correlated with the poor remodeling activity of the bone graft.[33] These features indicate that periosteum should be considered to be a structure with regenerative capacity. This suggests the need to restore the essential osteogenic activity of periosteum on bone graft in combination with grafting of MB-MSCs. This approach assists in the early induction of a reparative response by an increase in the formation of a cortical shell around the grafted bone.[34,35] Agata et al.[34] have also shown that periosteal cells act as progenitor cells with the ability to proliferate and expand. Thus, periosteum-derived cells are another suitable source for bone tissue engineering.

Based on clinical observation, radiologic examination, histological analyses, and biomechanical measurements, the current study supports the essential role of periosteum in the process of bone repair. In addition, the regenerative effect of combining BM-MSCs with periosteum showed better outcomes in both the quantity and quality as compared to BM-MSCs alone. Furthermore, the MB-MSCs used in the current study are derived from an allogenic source, which is more convenient for isolation and expansion when compared with periosteum-derived cells. To further enhance current bone tissue engineering strategies, a successful cellular replacement for periosteum or tissue-engineered periosteum should be investigated. Zhang et al.[11] previously reported successful regeneration of segmental bone defects in rabbit ulnas using periosteum encapsulated scaffolds seeded with MSCs, with an increase in the newly formed bone area to 80.1% ± 9.6%. This result is compatible with the results of the current study at 80.5% ± 4.96%.

Xenogeneic demineralized cancellous bone grafts, which have the advantages of favorable cellular compatibility and histocompatibility as a scaffold, have widely been used for the repair of short bony defects showing the induction of new bone formation and good mechanical properties. Osteoinductive structures in demineralized bone graft include a series of low-molecular-weight glycoproteins with bone morphogenetic proteins. These proteins promote chondroblastic differentiation of mesenchymal cells and create new bone formation via endochondral osteogenesis.[1,31,35] The bone formation process increases when decalcification of cortical bone exposes osteoinductive growth factors buried within the mineralized matrix. However, bone grafting has not been successful in the repair of large bone defects.[13] BM-MSCs, which can be seeded to the HDCB graft for construction of the tissue engineered bone graft, has been suggested as an effective option for reconstruction of large bone defects.

In the group repaired by periosteum-wrapped HDCB graft seeded with BM-MSCs, bone healing and union were significantly accelerated as compared to the other 3 groups. Increased density at the graft site and early fusion of cortical bone were observed. In addition to new bone formation demonstrated histologically, a significant amount of regenerated capillary vasculature between the new bones was also being observed in a high proportion of grafted bone pores. Zhang et al.[11] reported similar results when incorporating MSCs and periosteum-loaded poly scaffolds. However, our findings have notable differences from results of Zhang et al.,[11] as HDCB/BM-MSCs grafts were significantly superior to periosteum-wrapped HDCB grafts in terms of union rates and capillary density.

For improved biochemical analysis for bone regeneration, a three-point bending test should be performed to evaluate the degree of scaffold integration with the host bone.

In conclusion, this study demonstrates that repair of bone defect in a rabbit model can be achieved through bone grafting using BM-MSCs implanted on a xenogeneic demineralized cancellous bone scaffold. New bone formation was optimized with preservation of the periosteum at the site of injury. The combination of biocompatible material, the ability for self-renewal, differentiation of mesenchymal stem cells with the augmenting effects of periosteum may prove to be an extremely promising approach in the fields of orthopaedic and plastic surgery.